Genomic characterization of Ralstonia solanacearum phage phiRSA1 and its related prophage (phiRSX) in strain GMI1000

RSA1 is a wide-host-range bacteriophage isolated from Ralstonia solanacearum. In this study, the complete nucleotide sequence of the RSA1 genomic DNA was determined. The genome was 38,760 bp of double-stranded DNA (65.3% G+C) with 19-bp 5′-extruding cohesive ends (cos) and contained 51 open reading frames (ORFs). Two-thirds of the RSA1 genomic region encodes the phage structural modules, and they are very similar to those reported for coliphage P2 and P2-like phages. A RSA1 minireplicon with an 8.2-kbp early-expressing region was constructed. A late-expression promoter sequence motif was predicted for these RSA1 genes as 5′ TGTTGT-(X)₁₃-ACAACA. The genomic sequence similarity between RSA1 and related phages 52237 and CTX was interrupted by three AT islands, one of which contained an insertion sequence element, suggesting that they were recombinational hot spots. RSA1 was found to be integrated into at least three different strains of R. solanacearum, and the chromosomal integration site (attB) was identified as the 3′ portion of the arginine tRNA(CCG) gene. In the light of the RSA1 gene arrangement, one possible prophage sequence previously detected on the chromosome of R. solanacearum strain GMI1000 was characterized as a RSA1-related prophage (designated RSX). RSX was found to be integrated at the serine tRNA (GGA) gene as an att site, and its size was determined to be 40,713 bp. RSX ORFs shared very high amino acid identity with their RSA1 counterparts. The relationships and evolution of these P2-like phages are discussed.